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D R Helinski

Publications and source records attributed to D R Helinski.

At least 91 records · Page 5Linked to original sources
PubMed1985

Deletion analysis of Rhizobium meliloti symbiotic promoters.

Previous examination of DNA sequences located 5' to Rhizobium meliloti nif transcription units has shown that extensive sequence homology exists among them. Here we have examined these reiterated sequences for their role in symbiotic gene regulation. Promoter deletion analysis has shown that although an extensive upstream DNA sequence 9160 bp) is required for full heterologous activation of the R. meliloti nifHDK promoter by the Klebsiella pneumoniae nifA protein in Escherichia coli, this region is not required for expression of R. Meliloti nif promoters in root nodules from plants grown under greenhouse conditions. In addition, a minimum functional symbiotic promoter sequence is defined, and a DNA sequence difference affecting regulation of two symbiotic promoters is discussed.

Bacterial Proteins↗
PubMed1985

DNA segments of the IncX plasmid R485 determining replication and incompatibility with plasmid R6K.

The expression of incompatibility properties between the IncX plasmids R6K and R485 of Escherichia coli was examined. For small autonomously replicating derivatives of both plasmid elements, the requirements for incompatibility expression include a functional R485 replicon and an active R6K beta-origin region. Functional R6K alpha and gamma origins are not directly involved in incompatibility expression between R6K and R485. A trans-acting replication system was constructed for plasmid R485. It consists of a 3.2-(kb) DNA fragment of R485 that specifies a product(s) in trans which supports replication from an R485 origin plasmid. A minimal R485 origin region of 591 bp was derived utilizing this trans-acting replication system and the nucleotide sequence of this origin region determined. The most striking feature of the sequence is the presence of six tandem 22-bp nucleotide sequence direct repeats.

Base Sequence↗
PubMed1985

Transcription signals in a region essential for replication of plasmid R6K.

A DNA fragment encoding for the enzyme chloramphenicol acetyltransferase (CAT) but lacking the CAT promoter sequence was used to probe transcription signals in a DNA fragment (640 base pairs) from plasmid R6K that plays a central role in R6K DNA replication. The R6K DNA fragment analyzed contains the seven 22-bp direct repeats which express R6K incompatibility and are required in cis for activity of all three of the R6K origins of replication. In addition to the previously identified promoter sequences for the R6K initiation protein pi (P pi), a relatively weak transcriptional promoter upstream to P pi was detected. On the opposite strand an efficient (94%) transcriptional terminator was identified. This terminator is located upstream to nucleotide sequences active in the repression of R6K DNA replication.

Acetyltransferases↗
PubMed1985

Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli.

A cDNA library was constructed from firefly (Photinus pyralis) lantern poly(A)+ RNA, using the Escherichia coli expression vector lambda gt11. The library was screened with anti-P. pyralis luciferase (Photinus luciferin:oxygen 4-oxidoreductase, EC 1.13.12.7) antibody, and several cDNA clones expressing luciferase antigens were isolated. One clone, lambda Luc1, contained 1.5 kilobase pairs of cDNA that hybridized to a 1.9- to 2.0-kilobase band on a nitrocellulose blot of electrophoretically fractionated lantern RNA. Hybridization of the cloned cDNA to lantern poly(A)+ RNA selected an RNA that directed the in vitro synthesis of a single polypeptide. This polypeptide comigrated with luciferase on NaDodSO4/PAGE and produced bioluminescence upon the addition of luciferin and ATP. A 1.8-kilobase-pair cDNA was isolated by probing the firefly cDNA library with the cDNA from lambda Luc1. This cDNA contained sufficient coding information to direct the synthesis of active firefly luciferase in E. coli.

Animals↗
PubMed1985

Mutations in direct repeat sequences and in a conserved sequence adjacent to the repeats result in a defective replication origin in plasmid R6K.

Plasmid pMM3 is a pBR322 derivative carrying the gamma origin of replication of the naturally occurring plasmid R6K. We have produced a gamma-origin mutant bank of this plasmid using the single-strand-specific mutagen sodium bisulfite. Members of this bank contain single or multiple mutations in the seven direct repeats and the flanking sequences in the gamma origin. Three mutants with defective gamma origins have been isolated from this mutant bank. Two of these direct repeat mutants, gamma 117 and gamma 120, are unable to replicate and also have lost the ability to bind the R6K initiation protein pi in vitro at one of the seven 22-base-pair direct repeats within their respective origins. Precise deletion of the damaged repeat of either of these mutants restores origin function, suggesting that the primary defect of these mutants involves a disruption of the normal spacing of pi binding and flanking sequences within the gamma origin. The third mutant, gamma 111, binds pi normally but replicates at a greatly reduced copy number due to a mutation near the seventh repeat. This mutation falls within a short sequence that appears to be conserved among a number of other plasmids that contain direct repeats within their origins of replication.

Base Sequence↗
PubMed1985

Comparison of 10 IncP plasmids: homology in the regions involved in plasmid replication.

We have examined the DNA homology in the replication regions of 10 IncP plasmids independently isolated from several different countries. Two regions of RK2, the best-studied plasmid of this group, are required for vegetative DNA replication: the origin of replication (oriV) and the trfA region, which codes for a gene product necessary for replication. Six of nine IncP plasmids studied were identical to RK2 in the oriV and trfA regions as shown by Southern hybridization. Three P plasmids, R751, R772, and R906, showed weaker homology with the RK2 trfA, region and hybridized to different-sized HaeII fragments than the other six plasmids. R751, R772, and R906 hybridized to the region of the RK2 replication origin which expresses P incompatibility but differed markedly from RK2 and the other six plasmids in the GC-rich region of the origin required for replication. These data indicate that the P-group plasmids can be divided into two subgroups: IncP alpha, which includes the RK2-like plasmids, and IncP beta which includes the R751-like plasmids.

DNA Replication↗
PubMed1985

Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria.

The replication and maintenance properties of the broad-host-range plasmid RK2 and its derivatives were examined in nine gram-negative bacterial species. Two regions of RK2, the origin of replication (oriV) and a segment that encodes for a replication protein (trfA delta kilD, designated trfA*), are sufficient for replication in all nine species tested. However, stable maintenance of this minimal replicon (less than 0.3% loss per generation under nonselection conditions) is observed only in Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, and Azotobacter vinelandii. Maintenance of this minimal replicon is unstable in Rhizobium meliloti, Agrobacterium tumefaciens, Caulobacter crescentus, Acinetobacter calcoaceticus, and Rhodopseudomonas sphaeroides. A maintenance function has been localized to a 3.1-kilobase (kb) region of RK2 encoding three previously described functions: korA (trfB korB1 korD), incP1-(II), and korB. The 3.1-kb maintenance region can increase or decrease the stability of maintenance of RK2 derivatives dependent on the host species and the presence or absence of the RK2 origin of conjugal transfer (oriT). In the case of A. calcoaceticus, stable maintenance requires an RK2 segment that includes the promoter and the kilD (kilB1) functions of the trfA operon in addition to the 3.1-kb maintenance region. The broad-host-range maintenance requirements of plasmid RK2, therefore, are encoded by multiple functions, and the requirement for one or more of these functions varies among gram-negative bacterial species.

Base Sequence↗
PubMed1984

Chromogenic immunodetection of human serum albumin and alpha-L-fucosidase clones in a human hepatoma cDNA expression library.

A human hepatoma cDNA library was constructed in lambda gt11, a bacteriophage vector that was designed to express cDNA-encoded antigenic determinants in Escherichia coli. The cDNA expression library contained approximately 8 X 10(6) recombinant phages with an average insert size of 780 bp. The feasibility of using a chromogenic immunodetection procedure to isolate cDNA clones was proved by isolating a human serum albumin (HSA) cDNA clone. An approximately 1.0-kb cDNA clone was then isolated by screening the library with rabbit anti-human alpha-L-fucosidase antibodies. The identity of the alpha-L-fucosidase cDNA clone was confirmed by DNA sequence analysis and a comparison of the predicted amino acid sequence to the amino acid sequences of human alpha-L-fucosidase tryptic peptides.

Bacteriophage lambda↗
PubMed1984

Cloning and characterization of small circular DNA from Chinese hamster ovary cells.

Small polydisperse circular (spc) DNA was isolated and cloned, using BglII from Chinese hamster ovary (CHO) cells. The properties of 47 clones containing at least 43 different BglII fragments are reported. The majority of the clones probably contain entire sequences from individual spcDNA molecules. Most of the clones were homologous to sequences in CHO cell chromosomal DNA, and many were also homologous to mouse LMTK- cell chromosomal sequences. The majority of homologous CHO cell chromosomal sequences were repetitive, although a few may be single copy. Only a small fraction of cloned spcDNA molecules were present in every cell; most occurred less frequently than once in 15 cells. Localization studies indicated that at least a portion of spcDNA is associated with the nucleus in CHO cells.

Animals↗
PubMed1983

Structural properties of the beta origin of replication of plasmid R6K.

The beta origin of replication of plasmid R6K, one of three active R6K origins of replication, requires most or all of a 1962-base pair (bp) sequence for activity. The nucleotide sequence of a portion of this functional beta origin was determined in an earlier study (Stalker, D., Kolter, R., and Helinski, D. (1982) J. Mol. Biol. 161, 33-43). In this work, the sequence of the remaining portion of this 1964-bp segment was obtained. In addition to its activity as an origin of replication, this sequence also contains sufficient information for autonomous replication in Escherichia coli. A 277-bp region containing seven 22-bp direct repeats is present at one end of the beta origin segment (Stalker, D., Kolter, R., and Helinski, D. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 1150-1154) while the other end contains a 140-bp sequence that includes a relaxation complex site. The 277-bp direct repeat region is required for activity of the beta origin. The start of the beta origin of replication as mapped by electron microscopy (Crosa, J. (1980) J. Biol. Chem. 255, 11075-11077) lies approximately 1000 bp away from the 277-bp region. The pi structural gene, which makes up most of the sequence between the direct repeats and the beta origin, is required in cis for beta origin activity. The pi protein also is required for beta origin activity but can be provided in trans. The nucleotide sequence just beyond the pi structural gene and within or near the start of beta origin of replication contains an open reading frame for a 151-amino acid protein. Deletions ranging from 94 bp to 1590 bp were obtained within the 1964-bp beta origin region. In every case, the deletion results in loss of origin activity even when the deleted sequence plus adjacent regions are provided in trans. These observations suggest a requirement for a specific secondary structure over an extensive region for beta origin activity.

Base Composition↗
PubMed1983

Structural relationships among Rhizobium meliloti symbiotic promoters.

Symbiotic nitrogen fixation by Rhizobium meliloti requires the developmentally specific expression of certain bacterial genes. One set of these genes encodes the subunits of nitrogenase, the enzyme responsible for the reduction of atmospheric dinitrogen to ammonia, and another set consists of closely linked genes also essential for nitrogen fixation. Examination of promoter and probable regulatory regions for these gene sets has revealed extensive DNA sequence conservation for more than 160 bp upstream of the respective transcription start points. Three such promoter regions have been identified in the nitrogen fixation (nif) gene cluster of R. meliloti strain 102F34. Using one of these promoter regions as a hybridization probe, three additional sequences were found in the genome of this strain. The DNA of other R. meliloti strains and Rhizobium species were also examined for homology to the symbiotically regulated promoters of R. meliloti 102F34. DNA sequences homologous to these R. meliloti promoters were found among diverse rhizobia, and in at least some cases were associated with nif genes.

Base Sequence↗
PubMed1983

Replication of derivatives of the broad host range plasmid RK2 in two distantly related bacteria.

A 0.7-kb segment of the broad host range plasmid RK2 containing the replication origin of this plasmid will replicate in Escherichia coli and Pseudomonas putida when this segment is joined to a 1.8-kb region of RK2 designated traA*. The presence of another region of RK2, designated trfB, that previously was implicated in RK2 replication had no effect on the maintenance of the RK2 trfA*-oriV replicon in these two organisms. These observations indicate a requirement for a minimal account of information for replication of this broad host range plasmid in two distantly related bacteria.

DNA Replication↗
PubMed1983

Release of initiation control by a mutational alteration in the R6K pi protein required for plasmid DNA replication.

Plasmid pRK419, a derivative of the naturally occurring antibiotic resistance plasmid R6K, contains the pir gene that codes for the pi initiation protein and the beta and gamma replication origins of R6K. A mutation in plasmid pRK419, designated cos405, results in an elevated plasmid copy number in Escherichia coli growing at 42 degrees C and an even greater increase in copy number when the cells are shifted to 30 degrees C. This mutation was assigned to the structural gene for the pi protein on the basis of suppression of the mutant phenotype in E. coli when the wild-type pi protein is supplied in trans. Nucleotide sequence analysis of the cos405 mutant confirmed the pir gene location of the mutation and showed that this mutation results in a single amino acid substitution (glycine to aspartic acid) at the 81st position of the 305-amino acid pi protein. The properties of this mutant suggest that the pi protein plays a role in the negative control of the frequency of R6K initiation in addition to its requirement for the initiation of plasmid replication.

Bacterial Proteins↗
PubMed1983

Isolation and characterization of the recA gene of Rhizobium meliloti.

Interspecific complementation of an Escherichia coli recA mutant with plasmids containing a gene bank of Rhizobium meliloti DNA was used to identify a clone which contains the recA gene of R. meliloti. The R. meliloti recA protein can function in recombination and in response to DNA damage when expressed in an E. coli recA host, and hybridization studies have shown that DNA sequence homology exists between the recA gene of E. coli and that of R. meliloti. The isolated R. meliloti recA DNA was used to construct a recA R. meliloti, and this bacterium was not deficient in its ability to carry out symbiotic nitrogen fixation.

Bacterial Proteins↗